Effect Of Coreopsis Tinctoria Microcapsules On TDC Gene Expression And Tyramine Production In Smoked Horsemeat Sausages

Smoked horsemeat sausage is a kind of traditional Smoked and naturally fermented sausage produced in Xinjiang,and has high tyramine content.To improve the food safety of Smoked horsemeat sausage,based on Smoked horsemeat sausage,two tyramine producing strains of Enterococcus hirae N47 and Enterococcus durans N54 were screened from Smoked horsemeat sausage by high-performance liquid chromatography(HPLC)and polymerase chain reaction(PCR).Reverse transcription-quantitative polymerase chain reaction(RT-q PCR)was used to probe into the effect of Coreopsis tinctoria essential oil(CTEO)and Coreopsis tinctoria essential oil microcapsules(CTM)on the expression of TDC gene cluster related genes of E.hirae N47 and E.durans N54.Finally,HPLC was used to verify the inhibitory effect of Coreopsis Tinctoria Extract on tyramine accumulation before and after embedding,so as to provide a theoretical basis for improving the safety and quality of Smoked horsemeat sausage.The specific research contents and results are as follows:(1)Isolation and identification of tyramine producing bacteria with TDC gene from Smoked horsemeat sausage89 tyramine producing strains in Smoked horsemeat sausages were isolated by the method of single-layer culture and double-layer color development.Then,75 strains containing tyrosine decarboxylase(tyr DC)gene were obtained by PCR amplification with specific primers.Futher,tyramine producing ability of tyr DC gene strains were detected by HPLC.Finally,the two strongest tyramine producing strains were selected and identified as Enterococcus hirae(190.757 μg/m L)and Enterococcus durans(210.034 μg/m L).(2)Effect of Coreopsis Tinctoria Extract on tyramine production by Enterococcus hirae N47 and Enterococcus durans N54The minimum bactericidal concentration(MBC)and minimum inhibitory concentration(MIC)of CTEO and CTM were obtained by double dilution method with Enterococcus hirae N47 and Enterococcus durans N54;Then,RT-q PCR was used to analyze the expression of genes related to the TDC pathway of the tested bacteria under the action of CTEO and CTM;HPLC was used to detect the effect of CTM on tyramine production of the two strains.The results showed that both CTEO and CTM had strong antibacterial activity against the tested strains,and the inhibitory effect of CTM was more significant(P <0.05);the MIC of CTM against E.hirae N47 and E.durans N54 were 0.78 mg/m L and 1.56 mg/m L respectively;The CTM could significantly inhibit the expression of tyr DC and tyr P genes in TDC gene cluster(P<0.05),thus reducing tyramine accumulation.Additionally,when the dosage of CTM was MIC,the tyramine production of E.hirae N47 and E.durans N54 decreased respectively by 93.34% and 89.43%,compared with the tyrosine control group(L).To sum up,the CTM can inhibit tyramine production by inhibiting the growth of E.hirae N47 and E.durans N54 and reducing the gene expression in tyramine synthesis pathway.(3)Effect of CTEO and CTM on tyramine production during fermentation of Smoked horsemeat sausageE.hirae N47 and E.durans N54 were inoculated into Smoked horsemeat sausage,and the CTEO and the CTM were added.The moisture content,pH,thiobarbituric acid reactive substances(TBARS),microorganism,tyrosine decarboxylase gene expression and tyramine accumulation of Smoked horsemeat sausage were determined.The results showed that CTEO and CTM significantly reduced tyramine accumulation(P<0.05)by inhibiting the growth and expression of tyrosine decarboxylase(tyr DC)genes of E.hirae N47 and E.durans N54.The tyramine content in Smoked horsemeat sausage were reduced by65.03% and 75.81% respectively by adding CTEO and CTM.Therefore,adding CTM during the process of making Smoked horsemeat sausage can effectively reduce the production of tyramine and improve the safety of Xinjiang Smoked horsemeat sausage.